Infection of Eupatorium yellow vein geminivirus (EpYVV, formerly called tobacco leaf curl virus, TLCV) causes variegation in Eupatorium makinoi Kawahara et Yahara leaves. We examined changes in photosynthesis during leaf development to clarify what is the primary event when photosynthesis is suppressed in virus-infected E. makinoi leaves. The gas-exchange rate, leaf absorptance, chlorophyll (Chl) and nitrogen contents, leaf anatomy and chloroplast ultrastructure were compared between virus-infected and uninfected E. makinoi leaves at various developmental stages. These photosynthetic properties did not differ between infected and uninfected leaves when they were young. However, when expanded, infected leaves showed lower maximum quantum yield of photosynthetic CO₂ uptake in the incident photosynthetically active photon fluence rate (PPFR), which was attributed to their lower Chl contents. The Chla / b ratio was higher and the grana had fewer thylakoids in the infected leaves, which are features common to Chl b-deficient mutants that have defects in Chl synthesis. Our results suggested that, in E. makinoi leaves, EpYVV infection primarily impairs Chl biosynthesis. Possible mechanisms of the suppression of photosynthesis in E. makinoi leaves by virus infection are discussed.